Abstract

Background and Purpose— Early reperfusion using tissue-type plasminogen activator is the only therapeutic agent to treat focal cerebral ischemia with proven efficacy in patients. Nevertheless, novel insights into the pathophysiology of neurons, glial cells, and the fate of the endothelium after stroke call for the use of new strategies to improve stroke treatment alone or in combination with tissue-type plasminogen activator-induced thrombolysis. Unfortunately, despite the plethora of drugs that display clear beneficial effects in animal models of experimental ischemia, their subsequent use in clinical trials has proven disappointing. As such, one is forced to consider that new animal models of focal cerebral ischemia may be required before clinical evaluation of a new molecule.

Methods— In situ microinjection of purified murine thrombin was used to trigger a local clot formation in anesthetized mice. Cerebral blood velocity was measured continuously throughout the duration of the study. The efficiency of recombinant tissue-type plasminogen activator to induce thrombolysis and its subsequent effect on infarct volume were then measured.

The only approved drug for stroke treatment in the United States and Europe is the recombinant tissue-type plasminogen activator (rtPA; Actilyse; Boehringer Ingelheim) used as a thrombolic agent. Nevertheless, the use of rtPA is limited to a 3-hour time window after the onset of symptoms because of risks related to hemorrhage.1 In addition, in experimental models of cerebral ischemia, tPA was also reported to have some deleterious effects by promoting neurotoxicity.2–5 Altogether, only a small percent of patients with stroke (approximately 5%) will benefit from rtPA-induced thrombolysis. With one stroke occurring every 45 seconds in the United States and one stroke-related death every 3 minutes, it becomes urgent to develop new or combined strategies to combat this disease. Despite the efforts of the scientific community to address this question during the last decade, all of the therapeutic strategies validated in animal models have failed in humans with the exception of rtPA-induced thrombolysis. The reason for this conundrum is not clear, but it is well admitted that our present experimental models of stroke are either too severe or not reproducible enough to correctly evaluate the efficiency of putative neuroprotectants alone or combined with thrombolysis.

There are several stroke models that have been used in a variety of animal species involving mechanical occlusions by using electrocoagulation,6,7 a filament,8 or a ligature.9,10 Other workers have used a second type of model involving injection of autologous or heterologous preformed fibrin, blood clots,11,12,13 including microemboli,14 or in situ clot formation by using rose Bengal.15,16 Although these last models are closer to what happens in patients with stroke, they lack reproducibility and uniformity in the resulting infarct volume, the location of the lesion,17,18 and/or display a high degree of mortality19 rendering the statistical analysis of the data confusing (for review, 20). In addition, despite successful rtPA-induced reperfusion, it is surprising to note, in these models, opposite effects on infarct size depending on the extent to which the models reflect the contribution of fibrinolysis, blood–brain barrier alterations, or neurotoxicity.

The purpose of this study was to validate a reproducible mouse model of thromboembolic stroke in which a beneficial effect of tPA-induced reperfusion similar to that noted in humans was observed.

Materials and Methods

Animals

Male Swiss mice (25–30 g; Janvier, France) were housed in a temperature-controlled room on a 12-hour light/12-hour dark cycle with food and water ad libitum. Experiments were performed in accordance with French ethical laws (act no. 87–848; Ministère de l’Agriculture et de la Forêt) and European Communities Council Directives of November 24, 1986 (86/609/EEC) guidelines for the care and use of laboratory animals.

Surgical Procedures and Physiological Monitoring

Animals were deeply anesthetized with isoflurane 5% and, thereafter, maintained with 2.5% isoflurane in a 70%/30% mixture of NO2/O2. A catheter was inserted into the tail vein to allow the intravenous administration (200 μL) of saline or tPA (10 mg/kg). Mice were intubated and mechanically ventilated (ventilator NEMI Scientific Inc) at 180 breaths per minute. Rectal temperature was maintained at 37±0.5°C throughout the surgical procedure using a feedback-regulated heating system. In a separate group, a femoral artery was catheterized for monitoring of blood gases (pH, po2, and pco2) 5 and 30 minutes before and after the ischemia, respectively.

Clot Formation

Before the surgical procedure, a pipette was made with hematologic micropipettes (calibrated at 15 mm/μL; Assistent ref. 555/5; Hoecht, Sondheim-Rhoen, Germany) by using an electrophysiology puller (PC-10; Narishige). Thereafter, the micropipette was pneumatically filled with 1 μL of purified murine alpha-thrombin (approximately 1000 NIH units/mg; Sigma-Aldrich) by applying negative pressure.

Mice were placed in a stereotaxic device, the skin between the right eye and the right ear was incised, and the temporal muscle was retracted. A small craniotomy was performed, the dura was excised, and the middle cerebral artery (MCA) was exposed. The pipette was introduced into the lumen of the MCA and 1 μL of purified murine alpha-thrombin (0.75 UI) was pneumatically injected (by applying positive pressure with a syringe connected to the pipette through a catheter) to induce the formation of a clot in situ (Figure 1). The pipette was removed 10 minutes after the injection of alpha thrombin at which time the clot had stabilized.

Figure 1. Description of the model. A, Schematic description of the surgical approach. B, Reactions involved in the formation of a crosslinked fibrin clot. C, Photomicrographs and schematic drawing of the different steps of the surgical procedure leading to in situ thrombin injection into the MCA and subsequent clot formation.

Tissue-Type Plasminogen Activator-Induced Thrombolysis

To induce thrombolysis, tPA (10 mg/kg; Actilyse) was intravenously injected (tail vein, 10% bolus, 90% perfusion during 40 minutes) 20 minutes after the injection of alpha-thrombin. The control group received the same volume of saline under identical conditions.

Monitoring of Cerebral Blood Velocity by Laser Doppler Flowmetry

Cerebral blood velocity was determined by laser Doppler flowmetry using a fiberoptic probe (Oxford Optronix) glued to the skull in the MCA territory. Cerebral blood velocity was measured before the injection of alpha-thrombin (100% baseline) and throughout the duration of the experiment (75 minutes).

Measurement of Locomotor Activity

To evaluate neurological deficits induced by our model of in situ thromboembolic stroke, studies of motor activity were performed 24 hours before ischemia and 6 hours, 24 hours, 48 hours, and 72 hours postischemia. Briefly, mice were placed in individual acrylic chambers (30×20×20 cm) for 90 minutes and horizontal and vertical motor activities were recorded with an activity box (Imetronic, Pessac, France) as described elsewhere.21 The number and extent of horizontal and vertical movements was determined by breaks in movement-sensitive photobeams that were then converted into locomotor activity counts.

Assessment of Lesion Volume and Histology

After 24 hours, the mice were euthanized and the brains were removed and frozen in isopentane. Cryostat-cut coronal brain sections (20 μm) were stained with cresyl violet and analyzed with an image analyzer. For volume analysis, one section out of every 10 was stained and analyzed (covering the entire lesion). Regions of interest were determined through the use of a stereotaxic atlas for the mouse and an image analysis system (Scion Image) was used to measure the lesion corresponding to the nonstained area. To identify the presence of hemorrhage after tPA treatment (if any), a second set of cryostat-cut coronal brain sections (20 μm) were stained using the Perls’ Prussian blue method and counterstained with nuclear fast red to reveal iron overload. A positive hemorrhagic control was performed by introducing a needle into the cortical part of the brain during the surgery.

Histology of the Clots

In a separate group, coronal sections of brain fixed in formalin were taken in the injection site area. The brain tissue was processed, embedded in paraffin, and 4-μm thick sections were cut and stained with hematoxylin–eosin–saffron for further neuropathological analysis.

Data Analyses

Data are expressed as mean±SD and statistical analysis was performed with the Mann–Whitney U test. For behavioral studies, within-group comparisons were performed using the Friedman test; following a significant Fr value, post hoc comparisons were performed using the Wilcoxon signed ranks test.

Results

In all mice tested, physiological variables (temperature, pH, pco2, and po2) remained within the normal range throughout the observation period (Table 1).

Table 1. Physiological Parameters Measured in Control Mice Before and After In Situ Thromboembolic Clot Formation

Clot Formation

In the present model, we performed an in situ injection of purified murine thrombin directly into the middle cerebral artery at the bifurcation of its anterior and posterior branches through a thin glass pipette implanted across the vascular bed (Figure 1A, C). Thrombin cleaves fibrinopeptides A and B from the alpha and beta chains of fibrinogen and activates factor XIII to generate a crosslinked fibrin clot (Figure 1B). Immediately after thrombin injection, the formation of clot is observed (Figure 1C) leading to a rapid and dramatic decrease of brain perfusion in the area located downstream of the injection site. A clot was formed in all the animals operated on and remained in the same place throughout the observation period (up to 60 minutes). Collateral brain perfusion leads to a retrograde blood supply in the occluded artery. However, this observation was not correlated with significant changes in cerebral blood velocity. Indeed, thrombin-induced in situ clot formation was associated with a dramatic and sustained reduction (60 minutes, mean reduction of 40% to 50%; Figure 2A; Table 2) in the local cerebral blood velocity as measured by laser Doppler sonography.

To determine the histological nature of the clots formed, staining of brain coronal sections harvested 20 minutes after thrombin injection were taken from occluded animals (see “Methods”). As shown in Figure 3, clots are mainly formed of polymerized fibrin containing a low number of cells and platelets. Furthermore, no evidence of hemorrhage was observed during or after, the surgical procedure (examined after Perls’ Prussian blue staining; Figure 4) when compared with the positive control for hemorrhage induced by a mechanical lesion.

Figure 3. Histological analysis of the clot formed before and after rtPA-induced reperfusion. Hematoxylin–eosin–saffron (HES) staining. Photomicrographs (×40) show representative HES staining of the MCA performed in control and rtPA-treated mice.

Figure 4. Histological analysis of hemorrhage. Photomicrographs (×20) show a representative Perls’ staining performed in control and rtPA-treated mice. Positive controls for hemorrhage were performed by using a mechanical lesion.

Infarct Volume

Infarct volumes were measured in 10 mice. Figure 2B illustrates a set of stained slices from a representative untreated mouse. All animals showed infarction that was restricted to the cortex with a mean lesion volume of 36.6±8.9 mm3. The lesion volume was distributed from the anterior (position +3.2 from the bregma) to the posterior brain (−4.8 mm from the bregma) with a distribution that was homogeneous between animals (Figure 2C). Our data show that the present model is highly reproducible not only based on the final lesion volume, but also on the distribution of the brain areas that are damaged.

Twenty minutes after clot formation, mice were intravenously injected either with buffer (saline, n=10) or rtPA (Actilyse; 10 mg/kg; n=10) in the tail vein (10% bolus, 90% perfusion during 40 minutes). As illustrated in Figure 5A, rtPA treatment leads to the initiation of reperfusion approximately 15 minutes after the beginning of the injection and was complete 30 to 50 minutes after the onset of the treatment. Table 2 shows the mean value of cerebral blood velocity reduction for each group of animals (n=10). No significant difference in the reduction of blood velocity (40% to 50% reduction in the 2 groups) was observed after thrombin injection. rtPA treatment initiated 20 minutes after the occlusion leads to a significant and effective reperfusion 25 minutes later (ie, MCA occlusion +45 minutes), which remained stable with blood velocity being close to the initial preocclusion levels. Despite clear dissolution of the fibrin clot (Figure 3), no evidence of hemorrhage was observed under these conditions in any of the animals tested as was estimated by Perls’ staining at 24 hours postreperfusion (Figure 4). Figure 5B shows a set of representative brain sections taken from an rtPA-treated mouse. The extent of the final brain damage is homogeneous. All rtPA-treated mice showed brain infarction that was restricted to the cortex with a mean lesion volume of 23.1± 7.2 mm3 (n=10) with the same distribution as in control animals (Figure 5C).

Infarct volumes from 10 control mice and 10 rtPA-treated animals were compared. First, the ratio between each contralateral and ipsilateral hemisphere was calculated to estimate the presence of edema in both rtPA-treated animals and nontreated mice (Figure 6A). The analysis showed that no edema could be detected either in control or in rtPA-treated animals (0.95 for versus 0.93, respectively). The lesion volume was then measured by subtracting the volume of healthy tissue in the ipsilateral hemisphere from the total volume of the contralateral hemisphere (Hc-Hi in Figure 6B). The data show that rtPA administered 20 minutes after formation of the clot protects brain tissue with a reduction of the infarct size of approximately 36.8% when compared with control animals (P=0.0032) as measured at 24 hours postictus. As reported in clinical trials and as shown in the present mouse model of thromboembolic stroke, rtPA treatment is clearly beneficial.

Figure 6. rtPA-induced thrombolysis protects the brain against thromboembolic ischemia. A, Graphs of brain edema for control and rtPA-treated mice (ratio of the contralateral hemisphere (C) to the ipsilateral hemisphere (I) total volume, n=10). B, Graphs of the ischemic lesion of controls and rtPA-treated mice. The ischemic lesions were measured by subtracting the volume of healthy tissue of the ipsilateral hemisphere from the total volume of the contralateral hemisphere (Hc-Hi; n=10). Statistical analyses were performed using the nonparametric Mann–Whitney test. C, Graph of the locomotor activity measured 24 hours before ischemia and 6 hours, 24 hours, 48 hours, and 72 hours postischemia (n=5 animals per groups). The neurological deficit was evaluated from the basal activity (100%) measured 24 hours before ishcemia (#). The recovery was measured from the initial deficit induced by the embolic stroke (+6 hours) to +24 hours, 48 hours, and 72 hours (∗). Within-group comparisons were performed using the Friedman test; following a significant Fr value, post hoc comparisons were performed using the Wilcoxon signed ranks test.

Evaluation of Neurological Deficits and Functional Recovery

The horizontal and vertical motor activities were recorded with an activity box over 72 hours. rtPA-treated mice showed faster functional recovery than saline-treated mice. This recovery was significant at +24 hours, 48 hours, and 72 hours when compared with the initial neuronal deficit induced by the embolic stroke (n=5; P<0.05; Figure 6C).

Discussion

Based on the fact that thrombin is the main initiator and mediator of in situ clot formation and that rtPA is the only approved treatment for stroke, we developed a new model of thromboembolic stroke in mice. We described a mouse model of in situ thromboembolic stroke induced by the local injection of purified thrombin that results in reproducible ischemic brain damage and shows a significant improvement after rtPA-induced reperfusion. Several models of thromboembolic stroke have been developed in a variety of animal species. Nevertheless, the use of transgenic animals to further investigate mechanisms that control neuronal, glial, or endothelial outcome after stroke, including inflammatory processes, blood–brain barrier leakage or disruption, and neurotoxicity, requires the development of a reproducible and relevant model for stroke in mice.

We describe in the present study a model of embolic focal cerebral ischemia initiated by a fibrin-rich embolus formed in situ. The embolic focal cerebral ischemia models in rats and rabbits are induced by injection of a clot or clots through the extracranial segment of the internal carotid artery (ICA). Although there is a high probability that the MCA is the recipient of emboli, the location of infarcts in these models is not reproducible.11 This difficulty was overcome by a local delivery of an intact fibrin-rich embolus into the segment of the ICA near the origin of the MCA.12 Similar strategies were developed in mice by using injection of microemboli. However, in this later case, position and removal of an intraarterial catheter is a source of putative endovascular injuries22 and subsequent inflammatory processes. We provide an original strategy to initiate the cascade of events leading to the generation of an autologous thrombus directly into the MCA that does not require arterial catheterization and leads to a reproducible ischemic brain volume (36.6±8.9 mm3). Clot formation is associated with a decrease of the cerebral blood velocity to 40% to 50% of the preembolization levels in the ipsilateral MCA territory, a decrease that persists for at least 60 minutes after embolization.

Our model differs from existing models in several important ways. First, the clot is always located at the right place compared with other models that normally require the injection of a large number of preformed clots to promote MCA occlusion. These clots could lead to secondary occlusions in smaller vessels and subsequent variability of the ischemic damages measured, increasing also the degree of mortality. In contrast, the present model leads to a clot formation with accurate and reproducible brain damage. Although we cannot exclude the presence of secondary microthrombi in small arteries or capillaries, careful analysis of the brain sections did not reveal secondary thrombi nor were microinfarcts observed outwith the main ischemic lesion. These data suggest the presence of only one main thrombus, which is completely dissolved by rtPA. Second, our model is in agreement with the data obtained in clinical trials in which the beneficial effect of rtPA-induced thrombolysis is observed23 and thus provides an original and ad hoc animal model of “stroke” to test new generations of thrombolytic agents or combined strategies, including thrombolysis and neuroprotection. More importantly, in the present model of embolic stroke, there were no deaths reported in the 50 animals operated so far. In models of thromboembolic cerebral ischemia reported in the literature, the number of animals that die during the surgical procedure or the interval of time between the end of the surgery and the evaluation of brain damage is usually high.19 As such, mortality rates induce a further critical parameter for the evaluation of both drug safety and neuroprotection. Unfortunately, statistical analyses are usually performed without considering these animals.

To evaluate the relevance of an animal model, one needs to demonstrate, as observed in clinical trials,23 that rtPA treatment leads to a reduction of ischemic brain tissue. A number of studies have reported on the role of tPA in ischemic brain injury in mice. Using the filament model8 to promote MCA occlusion, tPA-deficient mice had smaller infarcts, suggesting that tPA may have detrimental effects after cerebral ischemia,2,24,25 including blood–brain barrier leakage26,27,28 and neurotoxicity.3,29,30 However, using the model of Longa et al,8 others authors showed that tPA-deficient mice had larger infarcts.22 In a photochemical vascular injury model, the outcome of tPA-deficient mice depended on the severity of injury.16 Finally, in a mouse model of microembolic stroke,14 tPA-deficient animals displayed a delayed dissolution of the cerebral emboli and an aggravated ischemic infarct. It is possible that differences in clot structure, dependent on the model used, may determine outcome of rtPA treatment.18 Thus, the beneficial effect of rtPA-induced thrombolysis in mice is questionable. Probably due to the difficulty to manage the thromboembolic stroke model in mice, most of the studies reported in the literature to test the efficiency of rtPA-induced thrombolysis were performed in rats and showed a clear beneficial effect. We provide a mouse model of thromboembolic stroke in which beneficial effects of rtPA-induced thrombolysis were observed.

Although our present model remains an animal model and cannot be compared with clinical trials, it seems to display all the features of a good model for the preclinical evaluation of stroke therapies.

Acknowledgments

Sources of Funding

This work was supported by grants from the INSERM, University of Caen-Basse Normandie, Regional Council of Lower Normandy, FP6-project DiMI-LSHB-CT-2005-512146, Foundation Paul Hamel.